The overall objective for this proposal is to gain insight on the mechanisms by which Natural Killer (NK) cells are regulated either by MHC or non-MHC molecules and the mechanisms leading to granule exocytosis. Evaluating the extent and nature of abnormalities in the signal transduction pathways resulting from Wiskott-Aldrich Syndrome Protein (WASP) deficiency will enable us to define the essentiality of the defective molecule in these pathways. Correction of the cytoskeletal and signaling defects upon retroviral transduction of the NK cells with the wild type normal WASP will identify the role played by this molecule in NK signaling. Such analysis may also uncover the interplay between other signaling molecules and WASP for their mutual activation to occur. Alternative pathways for granule exocytosis in cytolytic NK cells are likely to exist and evaluating defects in a single signaling molecule could provide such information. It is not known what form the NK synapse takes when activating signals outweigh the inhibitory signals. We will describe the role of NKG2D, 2B4 and NKp46 activating receptors in Neuroblastoma, especially identifying the differences in molecular events occurring at the synapse of tumor-cell killing versus the tumor-cell sparing NK clone. The kinetics of receptor-ligand interactions and SMAC formation in the cytolytic NK cell are likely to be fast and only the temporal analysis of the molecular events performed in real time as are proposed in these studies, will likely identify the sequence of molecular activation. FRET analysis will identify co-association of the interacting molecules from the mere physical co-localization. We will address these issues through the following specific aims: Specific aim 1: To characterize and compare the MHC regulated temporal and spatial organization of supramolecular activation clusters (SMAC) in the immune synapse of normal NK cells during interaction with HLA class I deficient target versus the target expressing only one self- HLA class I molecule. Specific aim 2: To determine the role of (WASP) in NK cell signaling pathway(s) mediating granule exocytosis. Specific aim 3: To characterize the temporal and spatial redistribution of 2B4, NKp46 and NKG2D in the SMAC during non-MHC mediated NK cell interactions with the autologous tumor target, Neuroblastoma and identify differences in recruitment of signaling molecules to the immune synapse of the tumor-killing versus the tumor-sparing NK cell by analyzing both the in vitro and in vivo conjugates. Such analysis will improve our understanding of the biology of cytolytic effector lymphocytes that could be of potential use in immunotherapy.